The present invention relates to methods and systems for directional, enhanced fluorescence from molecular layers, including its use in determining the presence or absence of a substance with a fluorescent component.
The invention is an improvement on the fluorescent assay method and apparatus described in U.S. Pat. No. 4,649,280, which issued Mar. 10, 1987 to Holland and Hall. Fluorescent assay systems typically employ optical detectors to detect the light emitted from a fluorescing substance. When light of a wavelength known to excite the fluorescent component of a particular substance is incident on an unknown sample, the presence (or absence) of the substance in the sample may be indicated by detecting the presence (or absence) of a fluorescent emission. Well known to the art is that the excitation and emission wavelengths of a fluorescent material are different.
Any methods of chemical analysis, including fluorescent assay systems, are improved by increasing the reliability of their results. U.S. Pat. No. 4,649,280 describes a method which increases the intensity of the fluorescent light emitted from the sample. This increase decreases the sensitivity required in the optical detector to determine the presence of a substance, which allows the detection of smaller quantity of a substance in a sample.
The method and apparatus described in the patent increased the light intensity of a fluorescing substance by employing an optical waveguide to generate a strong electromagnetic field in the vicinity of a film of fluorescent material, and particularly from the layers of molecules of the material attached to a wall of the optical waveguide. The excitation radiation which was incident on the fluorescent material was self-coupled to the waveguide to support the propagation of the modes which generated the strong field. This combination caused more intense fluorescence (i.e., intensity of the fluorescence) relative to that excited by radiation incident on conventional systems (i.e., fluorescent material coated on a glass slide). The strong field generated by the propagating modes was responsible for the increased fluorescence, and the increase was a function of the dimensions of the waveguide which supported the waveguide modes. The intensity of the fluorescence could be increased nearly 200 times that of conventional systems.
A number of problems were presented with the prior art systems.
The first problem was that the fluorescence emitted at a given wavelength from conventional systems and the system described in U.S. Pat. No. 4,649,280 was diffusely distributed about a normal to the flat surface of the fluorescent material. The result of this diffuse distribution was that most of the fluorescence emitted by the sample was not collected by the photodetectors. This diffuse distribution was compensated to some extent by the increased enhancement from the system described in U.S. Pat. No. 4,649,280; however the basic inefficient distribution for detection purposes remained. Since only a fraction of the fluorescence was detected, the prior art measurement techniques and systems were inherently less efficient than one that can make use of most or all of the fluorescence at a given wavelength, as provided by the present invention.
Another problem with conventional systems and the improved system of U.S. Pat. No. 4,649,280 was that they only determined the presence, absence and, to a limited degree, the concentration of a material in a sample. Also absent from these systems were other tests which would further indicate the presence or absence of a fluorescent material in a sample. Finally, if the fluorescent test was inconclusive, or if a check of the results was desired to increase the conclusiveness of the analysis, a completely separate method of analysis had to be used. This added expense to the analysis and was time consuming.
W. R. Holland et al., Optics Letters, Vol. 10, No. 8, pp. 414-416 (August 1985) also describes waveguide mode enhancement of molecular fluorescence, as described in the aforementioned patent, and ascribes the enhancement to near field interaction between the fluorescent molecules and the waveguide modal fields. A. M. Glass et al., Optics Letters, Vol. 5, No. 9, pp. 368-370 (September 1980), also reports the enhancement of fluorescent material deposited on a silver film.